Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis with an estimated two billion people currently infected worldwide and over nine million new cases detected annually worldwide. This is the largest killer of patients with HIV/AIDS. Current methods of diagnosis are complex, time-consuming, unreliable or unaffordable for most countries where the disease presents a significant public health threat. New diagnostics are critically needed to address the global tuberculosis burden and improve control programs. Our Phase I studies have clearly demonstrated the feasibility of developing a rapid and accurate test for tuberculosis. In collaboration with the Infectious Disease Research Institute, scientists at Chembio Diagnostic Systems, Inc. have identified a set of novel seroreactive antigens, designed several highly performing polyepitope fusion proteins, and applied the selected antigens in the Chembio's innovative immunoassay format, the Dual Path Platform (DPP(R)), with proven high accuracy of rapid serodiagnosis. We found that a DPP test prototype had a sensitivity of 81% and specificity of 95% in evaluations with serum samples obtained from culture-confirmed patients with active pulmonary tuberculosis from endemic regions in Brazil and Indonesia. In this Phase II application we propose to complete development and evaluation of the rapid diagnostic test for tuberculosis which will have required performance and operational characteristics. The specific aims are to: 1) optimize assay design, 2) determine diagnostic test performance, and 3) validate test production protocols in preparation for regulatory approval. We expect that the proposed DPP assay will demonstrate improved sensitivity and specificity over the existing rapid tests and will be applicable for expedited diagnosis of tuberculosis worldwide. PUBLIC HEALTH RELEVANCE: Tuberculosis remains one of the leading causes of mortality due to infectious disease worldwide. The current methods for diagnosis of tuberculosis are time-consuming, complex and laborious, and/or too expensive for routine use in resource-limited settings where most of patients live. The delayed or missed diagnosis leads to spreading of infection, progression of disease, and increased mortality. The research proposed here aims at developing a simple and rapid (20 minutes) point-of-care diagnostic test that would improve control programs by helping accurately detect new cases of tuberculosis, enabling timely antibiotic therapy, and preventing disease transmission. In addition to the visual reading, there will be an option for automated readout of the test result. The proposed assay will be fully validated and evaluated with serum samples from six countries representing various geographical areas and epidemiological setting associated with tuberculosis. These studies will demonstrate superior sensitivity and specificity as well as improved operational characteristics over the existing technologies.